Anticancer agents and methods of manufacture

ABSTRACT

A therapeutic metal coordination compound, particularly useful as an anticancer agent, which is the reaction product of a hydroxy quinone and a cisplatinum (II) compound substituted with chloro ligands and ammonia or derivatized ammonia ligands and the process for its manufacture.

BACKGROUND OF THE INVENTION

In previous work [Acta Pharmaceutica, Suecica, 15, 382-388 (1978),published February 1, 1979] applicant S. Yolles and co-workers V. Mortonand B. Rosenberg reported that composites of cisdichlorodiammine-platinum(II), abbreviated cis-Pta₂ Cl₂, withdoxorubicin and cyclophosphamide in poly (lactic acid), (PLA), showed asubstantial and unexpected improvement in the life span of mice, incomparison with composites containing only cis-Pta₂ Cl₂. Specifically,64 mg of composite cis-Pta₂ Cl₂ -doxorubicin-cyclophosphamide per kgmouse could be tolerated whereas cis-Pta₂ Cl₂ is toxic at 7 mg/kg mouse.

This work was covered by U.S. application Ser. No. 859,766 filed Dec.12, 1977, for "Controlled Release of Anticancer Agents fromBiodegradable Polymers" in the name of Seymour Yolles.

Complexes of 5,8-dihydroxy-1,4 naphthoquinone with several metals(beryllium, cobalt, copper, nickel and zinc) have been reported andtheir thermal stabilities determined [refer R. S. Bottei and P. L.Gerace, J. Inorg. Nucl. Chem., 23, 245, (1961)]. This study reported anunsuccessful attempt to prepare a platinum metal compound with5,8-dihydroxy-1,4-napthoquinone.

H. D. Coble and H. F. Holtzclaw, Jr., J. Inorg. Nucl. Chem., 36, 1049(1974) reported the properties of chelate polymers of copper (II) withseveral hydroxyquinones, including 5,8-dihydroxy-1,4 napthoquinone,1,4-dihydroxy-and 1,5-dihydroxy-9,10 anthraquinone and 6,11-dihydroxynaphthacenequinone.

More recently, C. G. Pierpont, L. C. Francesconi and D. M. Hendrickson,Inorg. Chem. 17, 3470 (1978) reported preparation and characterizationof binuclear nickel (II) and copper (II) complexes bridged by thedianions of 5,8-dihydroxy-1,4-naphthoquinone, and 1,4-dihydroxy-and 1,5dihydroxy-9,10 anthraquinones.

S. Cenini, R. Ugo and G. Lamonica, Jl. Chem. Soc [A], 1971, p. 416reported the preparation of two different chelates, with benzoquinone,one being of the 2-olefinic type and the other an orthoquinone, byreaction with diphosphine PtCl₂ (O).

M. Gonsalves, et al, Europ. J. Cancer, Vol. 14, p. 1185 (1978) disclosesthe preparation and therapeutic advantages of "Ouelamycin," a complex ofdoxorubicin (Adriamycin®) with iron having the formula: ##STR1##

Finally, the September, 1979, Am. Chem. Soc, InorganicAbstracts,Abstract 169, of Papers advises that F. T. Greenaway and J. C.Dabrowiak studied the metal-binding properties of doxorubicin and thechemically related compound, daunomycin, for Cu (II), Zn (II), Mg (II),Co (II), Ni (II) and Fe (III) ions.

According to the spectroscopic data, the metals bind to the aglyconeand, at least in the case of Cu (II), to the amine function ofdaunosamine.

The distinction between daunomycin and doxorubicin is seen from thefollowing structural formula: ##STR2## wherein R=H for daunomycin andR=OH for doxorubin

SUMMARY OF THE INVENTION

This invention relates broadly to a metal coordination compoundcomprising, as a therapeutic portion,

(a) a multi-ring quinone having at least one hydroxy group in a ringfused to the quinone ring as ligand,

(b) chelated at keto and hydroxy groups with the platinum metal

(c) in a cis-dichloro platinum (II) compound having two platinum metalcoordinate bonds each joined to a member of the group consisting ofammonia, mono-substituted ammonia and ammonia radicals connected one tothe other by simple valence bonds through adjacent carbons of a carbonring compound, and

(d) additional cis-dichloro platinum (II) compound not bonded to thehydroxy quinone but coordinated to said platinum which is bonded to saidhydroxy quinone, and, as a non-therapeutic portion,

(e) excess platinum in the form of tetrachloroplatinate anion, togetherwith the method of manufacturing such compounds.

DETAILED DESCRIPTION OF THE INVENTION

The anticancer properties of cis-dichlorodiammineplatinum (II),abbreviated cis-Pta₂ Cl₂, are well known, as are also the anticancerproperties of doxorubicin. However, cis-Pta₂ Cl₂ is relatively toxic,and the release rates of Pta₂ Cl₂ and doxorubicin, as individualcomponents of mixtures, such as taught in U.S. Application Ser. No.859,766 supra, can possibly vary independently one from the other indifferent utilization environments, contributing to a loss of sustaineddosage control.

I have now found that it is possible to prepare platinum metalcoordination compounds of multi-ring quinones having at least onehydroxy group in a ring fused to the quinone ring as ligand, whichdefinition also includes doxorubicin specifically.

Surprisingly, these metal coordination compounds, specifically thecompound doxorubicin with cis-dichlorodiammine platinum (II), displays asynergistic increase in anticancer activity as compared withdoxorubicin, cis-Pta₂ Cl₂ (II) mixtures per se. In addition, metalcoordination with a number of other hydroxy-substituted quinoneshereinafter described provides completely new anticancer agents notheretofore available.

The preparation of metal coordination compounds according to thisinvention cannot be effected by the usual techniques but, instead,requires prolonged special ball milling of mixtures of the specifichydroxy-substituted multi-ring quinones with the cis-dichloro diammoniatype platinum (II) compound in the presence of a liquid heat sink,preferably a solvent for at least one of the ingredients. Ball millingmust be conducted in ceramic or glass jars using glass, ceramic or othernon-metallic balls, the progress of coordination complexing beingindicated by the gradual development of an intense purple coloration ofthe reaction mixture.

Using quinizarin as example (refer Example 3 infra) it is postulatedthat the dihydroxy quinone metal coordination compounds according tothis invention have the following general structure, the therapeuticportion being bracketed whereas the non-therapeutic portion isrepresented by the tetrachloroplatinate anion shown to the right. Thecompound illustrated has a Pta₂ Cl₂ to quinone ratio of 11 to 2 whichagrees, within limits of experimental error, with the analyses made foridentified Examples infra. ##STR3##

The foregoing postulated structure is confirmed by the stoichiometricanalyses assumed and actually conducted for the several Exampleshereinafter described. Thus, in Examples 2, 3 and 4, the molar ratio ofcis Pta₂ Cl₂ to quinone approximates 5:1 or 6:1.

A somewhat simpler structure is postulated for the monohydroxy quinonejuglone of Example 1, this being ##STR4## which displays a ratio of cisPta₂ Cl₂ to quinone of approximately 3:1.

In order to obtain the necessary product compositions, the molar chargeproportions supplied to the ball-milling apparatus should be, in termsof Pt compound to quinone, 5:1 to 8:1 for dihydroxy quinones and 3:1 to5:1 for monohydroxy quinones.

In addition to those cited in the examples, the followinghydroxyquinones can be used to practice this invention: alizarin, 1,8dihydroxy-9,10-anthraquinone, purpurin, daunomycin, carminomycin,8,11-dihydroxy-5,12-naphthacenedione,1,4-dihydroxy-5,8-bis[2-(hydroxyethyl) amino ethyl]amino-9,10-anthracenedione.

The following are typical examples of compounds prepared according tothis invention, the platinum ingredient being dichlorodiammineplatinum(II), i.e., Pta₂ Cl₂ or dichloro 1,2-cyclohexanediaminoplatinum (II),##STR5##

EXAMPLE 1

Preparation of juglone cis-Pta₂ Cl₂ comples (I)

Juglone is a monohydroxy quinone having the structural formula: ##STR6##

Into a 120 ml. ceramic jar containing approximately 70 g. of glass balls(5 mm. in diameter) and 40 ml of DMF was added 0.5 g cis-Pta₂ Cl₂ and0.074 g. juglone and the mixture ball-milled at room temperature(approximately 104 r.p.m. for a 31/4" outside diameter ball mill jar andapproximately 170 r.p.m. for a 2" outside diameter ball mill jar) for120 hours. At the conclusion of ball-milling the reaction mixture had anintense blue coloration.

The ball-milled suspension was filtered and the filtrate evaporatedunder approximately 10 mm. Hg vacuum to dryness. The residue wasslurried in methylene chloride to remove unreacted juglone. Theprecipitate was collected on a frit-glass filter, redissolved indimethyl formamide (10-15 ml.) and to the clear filtrate was added anexcess of methylene chloride. The precipitate was collected andrecrystallized as above to give 0.59 g. of complex I as a purpleproduct.

The analysis calculated for C₁₀ H₂₂ N₆ Cl₆ Pt₃ O₃ was C, 11.16%; H,2.23%; N, 7.81%; Pt, 54.48%. The analysis actually found was C, 11.68%;H, 2.69%; N, 8.61%; Pt 55.51%. Absorption maxima (dimethyl formamide)was 270 and 380 nm. The ratio of cis-Pta₂ Cl₂ to juglone is 3:1.

EXAMPLE 2

Preparation of 5,8-dihydroxy-2-methyl-1,4 napthoquinone cis-Pta₂ Cl₂complex (II). 5,8-dihydroxy-2-methyl-1,4 naphthoquinone has thestructural formula: ##STR7##

Following the procedure hereinbefore described for Example 1, 5,8dihydroxy-2-methyl-1,4 naphthoquinone (0.085 g.) and cis-Pta₂ Cl₂ (0.50g.) were condensed to give 0.43 g. of product II as purple crystals.

Analysis calculated for C₁₁ H₃₈ N₁₀ Pt₅ O₄ Cl₁₀ was C, 7.76%; H, 2,25%;N, 8.23%; Pt, 57.23%. The analysis actually found was C, 7.65%; H,3.08%; N, 7.30%; Pt, 56.31%. Absorption maxima in dimethylformamide was270 mm., 410 nm.

The ratio of cis-Pta₂ Cl₂ to the quinone approximated 5:1.

EXAMPLE 3

Preparation of 1,4 dihydroxy-9, 10-anthraquinone cis-Pta₂ Cl₂ complex(III).

1, 4-dihydroxy-9, 10 anthraquinone (quinizarin) has the structuralformula: ##STR8##

Following the procedure hereinbefore described for Example 1, 1, 4dihydroxy-9, 10-anthraquinone (0.20 g.) was reacted with cis-Pta₂ Cl₂(0.50 g.) to give 0.34 g. of product III as purple crystals.

Analysis calculated for C₁₄ H₄₄ N₁₂ Cl₁₂ Pt₆ O₄ was C, 8.26%; H, 2.16%;Cl, 20.65%; Pt, 57.52%. The analysis actually found was C, 8.68%; H,2.48%; Cl, 20.10%; Pt, 56.76%.

Absorption maxima in dimethylformamide are 275, 525, and 565 nm.

The ratio of cis-Pta₂ Cl₂ to quinizarin in the compound approximates6:1.

EXAMPLE 4

Preparation of doxorubicin cis-Pta₂ Cl₂ complex (IV)

Doxorubicin has the structural formula: ##STR9##

First, doxorubicin free base was prepared from doxorubicinhydrochloride.

Doxorubicin hydrochloride was converted to free base by bubbling gaseousammonia through a capillary pipet into a stirred suspension ofdoxorubicin hydrochloride (0.050 g.) in 15 ml. of an 8:1 mixture ofchloroform and methanol at 0° C. The bubbling of ammonia and thestirring were continued until the mixture was red-orange. After theammonium chloride settled out overnight, the mixture was filtered andthe filtrate, after dilution with 150 ml. of the same chloroform,methanol solvent supra, was heated at 60° C. for one hour to give asolution of 0.037 g. of doxorubicin base in 100 ml. of solvent.

Following the procedure hereinbefore described for Example 1,doxorubicin base (0.037 g.) and cis-Pta₂ Cl₂ (0.104 g.) were condensedto give 0.103 g. of product IV as purple crystals.

Analysis calculated for C₂₇ H₆₅ O₅ N₁₁ Cl₈ Pt₅ was C, 15.85%; H, 2.89%;N, 7.53%; Pt, 47.70%. The analysis actually found was C, 15.99%; H,3.19%; N, 7.65%; Pt, 47.62%. Absorption maxima in dimethylformamide was475, 530, 575 nm.

The ratio of cis-Pta₂ Cl₂ to doxorubicin approximates 5:1.

EXAMPLE 5

Preparation of 5, 8-dihydroxy-1,4-naphthoquinone (naphthazarin) cis-Pta₂Cl₂ complex (V).

Following the procedure hereinbefore described for example 1,naphthazarin (0.0386 g.) and cis-Pta₂ Cl₂ (0.3673 g.) were condensed in40 mlDMF to give 0.32 g. of recrystallized product (V) as dark bluecrystals.

Absorption maxima in dimethylformamide was 301, 271, 606, 563.

EXAMPLE 6

Preparation of quinizarin cis-dichloro-1,2-cyclohexane diamino platinumII.

Following the procedure hereinbefore described for example 1, quinizarin(0.0447 g.) and cis-dichloro 1,2 diaminocyclohexane (0.3692 g.) werecondensed in 40 ml. of DMF to give 0.30 g. of product VI as purplecrystals.

Absorption maxima in dimethyl formamide was 303, 226, 565, 533.

Table I is a summary of the spectroscopic data identifying the severalproducts of Example 1-6, inclusive.

                                      TABLE I                                     __________________________________________________________________________    Ultraviolet Maxima (log εmax)                                                             Reagent maxima                                                                         Reagent maxima                                                                         New maxima                                             Example                                                                            present in                                                                             absent in                                                                              in complex                                             Number                                                                             complex (nm)                                                                           complex (nm)                                                                           only (nm)                               __________________________________________________________________________    cis Pt/Juglone 1    270      425      485                                     complex                      370(w)   380                                     cis Pt/Naphthazarin                                                                          5    301(sh)  546      606                                     complex             271      510      563                                     cis Pt/5,8-dihydroxy-2-methyl-                                                               2    303(sh)  358      303(shoulder)                           1,4 naphthaquinone  270(3.0) 370(w)                                           complex                                                                       cis Pt/Quinizarin                                                                            3    262      468      567(4.0)                                complex                      370(w)   525(4.1)                                cis Pt/doxorubicin complex (IV)                                                              4    495      530      575                                                         475                                                       cis diaminocyclohexane                                                                       6    303(sh)  468      565                                     dichloro Pt(II)/Quinizarin                                                                        266               533                                     complex                                                                       __________________________________________________________________________    Infrared Spectra Major Peaks (cm.sup.-1)                                                                      Reference and                                 Compound    Absorption cm.sup.-1                                                                     Assignment                                                                             Comments                                      __________________________________________________________________________    cis Pt/Quinizarin                                                                         3280       ν(NH.sub.3)                                                                         3                                             complex     3200       ν(NH.sub.3)                                                                         3                                             Example 3   2910       Chelated OH                                                                            1                                                         1650       δ NH.sub.3                                                                       3                                                         1605       C═O      4, shifted                                                                to lower wavelength                                                           upon metal chelation                          cis Pt/Doxorubicin                                                                        3290       ν(NH.sub.3)                                                                         3                                             complex     1750                                                                          1620- 1650 C═O                                                Example 4   1050-1150  C--O--Pt 5                                                         780-790                                                                        610                                                                           450       Pt--O    4                                             __________________________________________________________________________     References-                                                                   1. D. Hadzi, N. Sheppard, Trans. Far. Soc. 50, 911 (1954).                    2. R. H. Thompson, Naturally Ocurring Quinones, Academic Press. pp. 39-92     (1971).                                                                       3. H. Poulet, et al., Spectrochim Acta 20, 1855 (1964).                       4. K. Nakomoto, S. J. McCarthy, Spectroscopy and Structure of Metal           Chelate Compounds, Wiley 268-276 (1968).                                      5. L. J. Bellamy, Infrared Spectroscopy of Complex Molecules, Wiley           (1959).                                                                  

Table II is a summary of the results of in vivo testing of typicalproducts according to this invention. These experiments were performedin the standard assay of the Ascites Sarcoma 180 in the ICR mice. Therewere six mice in each cage, with two cages for the negative controls,two cages for the positive controls and one cage for each dose level ofdrug tested. Tumor cell injections of about 10⁶ cells were performed onDay 0 and drug injection Day 1, both given intraperitoneally (IP). Metalcoordination compounds were suspended in normal saline as a carrier.

The results of the in vivo tests performed on mice with two of thecompounds can be seen from Table II. These tests confirm the synergisticaction of the coordination compounds. Some doses were significantlybetter than the positive control (7 mg/kg single injection of cis-Pta₂Cl₂). The improvement in the life span compared to the positive controlis substantial. Larger doses of coordination compound over positivecontrol with reduced toxicity can be tolerated.

                  TABLE II                                                        ______________________________________                                        In vivo test results                                                                     Dose mg.   Average  %      No. of                                             compound/kg                                                                              day of   Increased                                                                            Cures                                   Compound   mouse      death    life span                                                                            (out of 6)                              ______________________________________                                        negative control                                                                         0          17.3     --     --                                      positive control                                                              7mg/kg cis Pta.sub.2                                                          Cl.sub.2   0          31.3     80     4                                       cis-Pt/Doxo com-                                                              pound Example 4                                                                          5          34.2     97     4                                                  10         36.0     108    6                                                  15         34.8     101    1                                                  20         26.2     51     1                                       negative control                                                                         0          13.8     --     --                                      positive control                                                              7mg/kg cis-Pta.sub.2                                                          Cl.sub.2   0          25       81     1                                       cis-Pt/Quiniz.                                                                Example 3  8          28       107    1                                                  12.5       26       88     0                                                  25         16.0     16     0                                                  50         2.75     -80                                                       100        3.8      -72                                            ______________________________________                                    

What is claimed is:
 1. A metal coordination compound comprising, as atherapeutic portion,(a) A multi-ring quinone having at least one hydroxygroup in a ring fused to the quinone ring as ligand, (b) chelated atketo and hydroxy groups with the platinum metal (c) in a cis-dichloroplatinum (II) compound having two platinum metal coordinate bonds eachjoined to a member of the group consisting of ammonia, mono-substitutedammonia and ammonia radicals connected one to the other by singlevalence bonds through adjacent carbons of a carbon ring compound, and(d) additional cis-dichloro platinum (II) compound not bonded to thehydroxy quinone but coordinated to said platinum which is bonded to saidhydroxy quinone, and, as a non-therapeutic portion, (e) excess platinumin the form of tetrachloro-platinate anion.
 2. A metal coordinationcompound according to claim 1 wherein said multi-ring quinone isdoxorubicin and said cis-dichloro platinum (II) compound iscis-dichlorodiammine platinum (II).
 3. A metal coordination compoundaccording to claim 1 wherein said cis-dichloro platinum (II) compound iscis-dichloro 1, 2 -cyclohexane diamino platinum (II).
 4. A metalcoordination compound according to claim 1 wherein said cis-dichloro(II) compound has the general formula cis (RNH₂)₂ dichloro platinum (II)wherein R can be hydrogen or an alkyl group having 1 to 10 carbon atoms.5. A metal coordination compound according to claim 4 wherein saidcis-dichloro platinum (II) compound is cis.
 6. A metal coordinationcompound according to claim 1 wherein said multi-ring quinone is amember of the group juglone, naphthazarin quinizarin, alizarin, and 8,11 dihydroxy 5, 12 naphthacenedione.
 7. The method of manufacturing ametal coordination compound of the composition of claim 1 comprising, inthe sequence recited, mixing said multi-ring quinone with saidcis-dichloro platinum II in a liquid heat sink comprising essentially asolvent for one of the reactants, ball milling said mixture with saidliquid heat sink at room temperature in a hermetically sealed ceramic orglass jar for at least 12 hours using glass, ceramic or othernon-metallic balls, filtering solids from said liquid heat sink and fromthe filtrate to recover said metal coordination compound.
 8. The methodof manufacturing a metal coordination compound according to claim 8wherein said solvent is a member of the group dimethyl formamide,dimethyl acetamide, methylene chloride, methyl pyrolidone, hexamethylphosphoramide.
 9. The method of manufacturing a metal coordinationcompound according to claim 7 wherein said cis-dichloro platinum (II)compound is proportioned to said quinones as charged in the ratios ofapproximately 5:1 to 8:1 in mols for quinones having two hydroxy groupsand approximately 3:1 to 5:1 in mols for quinones having one hydroxygroup.